Loss of p300 and CBP disrupts histone acetylation at the mouse Sry promoter and causes XY gonadal sex reversal.

CREB-binding protein (CBP, CREBBP, KAT3A) and its closely related paralogue p300 (EP300, KAT3B), together termed p300/CBP, are histone/lysine acetyl-transferases that control gene expression by modifying chromatin-associated proteins. Here, we report roles for both of these chromatin-modifying enzymes in mouse sex determination, the process by which the embryonic gonad develops into a testis or an ovary. By targeting gene ablation to embryonic gonadal somatic cells using an inducible Cre line, we show that gonads lacking either gene exhibit major abnormalities of XY gonad development at 14.5 dpc, including partial sex reversal. Embryos lacking three out of four functional copies of p300/Cbp exhibit complete XY gonadal sex reversal and have greatly reduced expression of the key testis-determining genes Sry and Sox9. An analysis of histone acetylation at the Sry promoter in mutant gonads at 11.5 dpc shows a reduction in levels of the positive histone mark H3K27Ac. Our data suggest a role for CBP/p300 in testis determination mediated by control of histone acetylation at the Sry locus and reveal a novel element in the epigenetic control of Sry and mammalian sex determination. They also suggest possible novel causes of human disorders of sex development (DSD).

The Gonadal Supporting Cell Lineage and Mammalian Sex Determination: The Differentiation of Sertoli and Granulosa Cells.

The supporting cell lineage plays a crucial role in nurturing the development of germ cells in the adult gonad. Sertoli cells in the testis support the progression of spermatogonial stem cells through meiosis to the production of motile spermatozoa. Granulosa cells, meanwhile, are a critical component of the ovarian follicle that produces the mature oocyte. It is a distinctive feature of the embryonic gonad that at least some of the supporting cells are derived from a single sexually bipotential precursor lineage. It is the commitment of this somatic lineage to either the Sertoli or granulosa cell fate that defines sex determination. In this chapter we review what is known about the key molecules responsible for this lineage decision in the developing mammalian gonads, relying primarily on data from studies of mice and humans. We focus on recent advances in our understanding of the mutually antagonistic interactions of testis- and ovary-determining pathways and their complexity as revealed by genetic analyses. For the sake of simplicity, we will deal with supporting cells in testis and ovary development in separate sections, but numerous points of contact exist between these accounts of gonadogenesis in male and female embryos, primarily due to the aforementioned mutual antagonisms. The final section will offer a brief synthesis of these organ-specific overviews and a summary of the key themes that emerge in this review of supporting cell differentiation in mammalian sex determination.

Genetic Analyses Reveal Functions for MAP2K3 and MAP2K6 in Mouse Testis Determination.

Testis determination in mammals is initiated by expression of SRY in somatic cells of the embryonic gonad. Genetic analyses in the mouse have revealed a requirement for mitogen-activated protein kinase (MAPK) signaling in testis determination: targeted loss of the kinases MAP3K4 and p38 MAPK causes complete XY embryonic gonadal sex reversal. These kinases occupy positions at the top and bottom level, respectively, in the canonical three-tier MAPK-signaling cascade: MAP3K, MAP2K, MAPK. To date, no role in sex determination has been attributed to a MAP2K, although such a function is predicted to exist. Here, we report roles for the kinases MAP2K3 and MAP2K6 in testis determination. C57BL/6J (B6) embryos lacking MAP2K3 exhibited no significant abnormalities of testis development, whilst those lacking MAP2K6 exhibited a minor delay in testis determination. Compound mutants lacking three out of four functional alleles at the two loci also exhibited delayed testis determination and transient ovotestis formation as a consequence, suggestive of partially redundant roles for these kinases in testis determination. Early lethality of double-knockout embryos precludes analysis of sexual development. To reveal their roles in testis determination more clearly, we generated Map2k mutant B6 embryos using a weaker Sry allele (Sry(AKR)). Loss of Map2k3 on this highly sensitized background exacerbates ovotestis development, whilst loss of Map2k6 results in complete XY gonadal sex reversal associated with reduction of Sry expression at 11.25 days postcoitum. Our data suggest that MAP2K6 functions in mouse testis determination, via positive effects on Sry, and also indicate a minor role for MAP2K3.

A novel mouse Fgfr2 mutant, hobbyhorse (hob), exhibits complete XY gonadal sex reversal.

The secreted molecule fibroblast growth factor 9 (FGF9) plays a critical role in testis determination in the mouse. In embryonic gonadal somatic cells it is required for maintenance of SOX9 expression, a key determinant of Sertoli cell fate. Conditional gene targeting studies have identified FGFR2 as the main gonadal receptor for FGF9 during sex determination. However, such studies can be complicated by inefficient and variable deletion of floxed alleles, depending on the choice of Cre deleter strain. Here, we report a novel, constitutive allele of Fgfr2, hobbyhorse (hob), which was identified in an ENU-based forward genetic screen for novel testis-determining loci. Fgr2hob is caused by a C to T mutation in the invariant exon 7, resulting in a polypeptide with a mis-sense mutation at position 263 (Pro263Ser) in the third extracellular immunoglobulin-like domain of FGFR2. Mutant homozygous embryos show severe limb and lung defects and, when on the sensitised C57BL/6J (B6) genetic background, undergo complete XY gonadal sex reversal associated with failure to maintain expression of Sox9. Genetic crosses employing a null mutant of Fgfr2 suggest that Fgr2hob is a hypomorphic allele, affecting both the FGFR2b and FGFR2c splice isoforms of the receptor. We exploited the consistent phenotype of this constitutive mutant by analysing MAPK signalling at the sex-determining stage of gonad development, but no significant abnormalities in mutant embryos were detected.

Characterising novel pathways in testis determination using mouse genetics.

Mammalian sex determination is the process by which the bipotential embryonic genital ridge commits to either the testicular or ovarian fate. The existence of a gonad primordium with 2 possible developmental outcomes, depending on the presence or absence of the Y chromosome, remains a fascinating paradigm for examining the manner in which genes cooperate to establish cell fate and regulate differentiation. Development of the mouse gonad offers a unique model for the investigation of sex determination, characterised by powerful transgenic, genomic and other experimental resources for the study of the molecular control of organogenesis. This review focuses on recent progress in our understanding of mouse testis determination, with an emphasis on multi-locus studies of mutant alleles aimed at constructing pathways of interacting gene products. Studies in 2 broad areas have been especially revealing: (i) identification of pathways required for the appropriate expression of Sry, and (ii) characterisation of the antagonistic interactions between the core testis- (SRY-SOX9-FGF9) and ovary- (RSPO1-WNT4-CTNNB1-FOXL2) determining gene regulatory networks. We discuss these advances with an eye on emerging themes in mammalian sex determination.

Transgenic expression of Map3k4 rescues T-associated sex reversal (Tas) in mice.

Disorders of sex development in the human population range in severity from mild genital defects to gonadal sex reversal. XY female development has been associated with heterozygous mutations in several genes, including SOX9, WT1 and MAP3K1. In contrast, XY sex reversal in mice usually requires complete absence of testis-determining gene products. One exception to this involves T-associated sex reversal (Tas), a phenomenon characterized by the formation of ovotestes or ovaries in XY mice hemizygous for the hairpin-tail (T(hp)) or T-Orleans (T(Orl)) deletions on proximal mouse chromosome 17. We recently reported that mice heterozygous for a null allele of Map3k4, which resides in the T(hp) deletion, exhibit XY ovotestis development and occasional gonadal sex reversal on the sensitized C57BL/6J-Y(AKR) (B6-Y(AKR)) genetic background, reminiscent of the Tas phenotype. However, these experiments did not exclude the possibility that loss of other loci in the T(hp) deletion, or other effects of the deletion itself, might contribute to Tas. Here, we show that disruption to Sry expression underlies XY gonadal defects in B6-Y(AKR) embryos harbouring the T(hp) deletion and that a functional Map3k4 bacterial artificial chromosome rescues these abnormalities by re-establishing a normal Sry expression profile. These data demonstrate that Map3k4 haploinsufficiency is the cause of T-associated sex reversal and that levels of this signalling molecule are a major determinant of the expression profile of Sry.

Gadd45γ and Map3k4 interactions regulate mouse testis determination via p38 MAPK-mediated control of Sry expression.

Loss of the kinase MAP3K4 causes mouse embryonic gonadal sex reversal due to reduced expression of the testis-determining gene, Sry. However, because of widespread expression of MAP3K4, the cellular basis of this misregulation was unclear. Here, we show that mice lacking Gadd45γ also exhibit XY gonadal sex reversal caused by disruption to Sry expression. Gadd45γ is expressed in a dynamic fashion in somatic cells of the developing gonads from 10.5 days postcoitum (dpc) to 12.5 dpc. Gadd45γ and Map3k4 genetically interact during sex determination, and transgenic overexpression of Map3k4 rescues gonadal defects in Gadd45γ-deficient embryos. Sex reversal in both mutants is associated with reduced phosphorylation of p38 MAPK and GATA4. In addition, embryos lacking both p38α and p38ß also exhibit XY gonadal sex reversal. Taken together, our data suggest a requirement for GADD45γ in promoting MAP3K4-mediated activation of p38 MAPK signaling in embryonic gonadal somatic cells for testis determination in the mouse.

Gene expression profiling reveals new potential players of gonad differentiation in the chicken embryo.

BACKGROUND: In birds as in mammals, a genetic switch determines whether the undifferentiated gonad develops into an ovary or a testis. However, understanding of the molecular pathway(s) involved in gonad differentiation is still incomplete. METHODOLOGY/PRINCIPAL FINDINGS: With the aim of improving characterization of the molecular pathway(s) involved in gonad differentiation in the chicken embryo, we developed a large scale real time reverse transcription polymerase chain reaction approach on 110 selected genes for evaluation of their expression profiles during chicken gonad differentiation between days 5.5 and 19 of incubation. Hierarchical clustering analysis of the resulting datasets discriminated gene clusters expressed preferentially in the ovary or the testis, and/or at early or later periods of embryonic gonad development. Fitting a linear model and testing the comparisons of interest allowed the identification of new potential actors of gonad differentiation, such as Z-linked ADAMTS12, LOC427192 (corresponding to NIM1 protein) and CFC1, that are upregulated in the developing testis, and BMP3 and Z-linked ADAMTSL1, that are preferentially expressed in the developing ovary. Interestingly, the expression patterns of several members of the transforming growth factor ß family were sexually dimorphic, with inhibin subunits upregulated in the testis, and bone morphogenetic protein subfamily members including BMP2, BMP3, BMP4 and BMP7, upregulated in the ovary. This study also highlighted several genes displaying asymmetric expression profiles such as GREM1 and BMP3 that are potentially involved in different aspects of gonad left-right asymmetry. CONCLUSION/SIGNIFICANCE: This study supports the overall conservation of vertebrate sex differentiation pathways but also reveals some particular feature of gene expression patterns during gonad development in the chicken. In particular, our study revealed new candidate genes which may be potential actors of chicken gonad differentiation and provides evidence of the preferential expression of BMPs in the developing ovary and Inhibin/Activin subunits in the developing testis.

First evidence of bone morphogenetic protein 1 expression and activity in sheep ovarian follicles.

Bone morphogenetic protein (BMP) 1 is a vertebrate metalloproteinase of the astacin family. BMP1 plays a key role in regulating the formation of the extracellular matrix (ECM), particularly by processing the C-propeptide of fibrillar procollagens. BMP1 also promotes BMP signaling by releasing BMP signaling molecules from complexes with the BMP-antagonist chordin. As a result of BMP1's dual role in both ECM formation and BMP signaling, we hypothesized that BMP1 could play a role in ovarian physiology. Using the sheep ovary as a model system, we showed that BMP1 was expressed in the ovary throughout early fetal stages to adulthood. Furthermore, in adult ovaries, BMP1 was expressed along with chordin, BMP4, and twisted gastrulation, which together form an extracellular regulatory complex for BMP signaling. Within ovine ovaries, immunohistochemical localization demonstrated that BMP1 was present in granulosa cells at all stages of follicular development, from primordial to large antral follicles, and that the levels of BMP1 were not affected by the final follicle selection mechanism. In cultured granulosa cells, BMP1 expression was not affected by gonadotropins, but BMP4 and activin A had opposing effects on the levels of BMP1 mRNA. BMP1 appeared to be secreted into the follicular fluid of antral follicles, where it is able to exert procollagen C-proteinase and chordinase activities. Interestingly, BMP1 activity in follicular fluid decreased with follicular growth.